Method for acyltransferase reaction using acyl coenzyme A

ABSTRACT

The present invention relates to a method for acyltransferase reaction in which an acyl group of acyl coenzyme A is transferred to an acyl group receptor characterized in that the reaction is carried out by production and/or reproduction of acyl coenzyme A from coenzyme A in a reaction system by a chemical thioester exchange reaction with acylthioester. The present invention, wherein expensive acyl CoA is reproduced nonenzymatically in a reaction system, enables to continuously carry out acyltransferase reaction only by putting a small amount of acyl CoA with a donor and a receptor of an acyl group into a system. Accordingly, the method of the present invention can be applied to an industrial production method of various kinds of compounds including useful biological molecules and synthesis of polymers such as polyester.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.10/542,733, filed Jul. 20, 2005 now U.S. Pat. No. 7,476,521; which is a371 of PCT/JP2004/000500 filed Jan. 21, 2004; the disclosures of each ofwhich are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a method for acyltransferase reactionfor transferring an acyl group to various organic compounds by acylcoenzyme A (hereinafter, coenzyme A may be referred to as CoA). Moreparticularly, it relates to a novel method for acyltransferase reactionin which the reaction is continuously carried out without additional useof a very expensive acyl CoA in a production process of an acyl compoundusing acyltransferase to significantly improve its productivity wherebyto enable acyltransferase to be utilized for industrial productionprocess of various compounds.

The present invention further relates to a CoA enzyme coupling methodwhere a chemical thioester exchange reaction is used as an acyl CoAreproduction system.

The present invention furthermore relates to a production process ofimportant physiologically active substances such as a sphingolipid usingCoA enzyme.

The present invention still further relates to a production process ofmacromolecular compounds by an enzymatic reaction. More particularly, itrelates to an efficient production process of biodegradablemacromolecular compounds, particularly polyester, where acyl coenzyme A(acyl CoA) is reproduced in a reaction system in the co-presence of athioester exchange reaction and an enzymatic polymerization reactionwhereby macromolecular compounds are able to be continuously synthesizedfrom thioesters.

BACKGROUND ART

CoA is a substance which functions as an acyl carrier/acyl activator inall biological species. For example, in addition to the fact that acetylCoA is a key substance for important biological metabolism of fattyacid, glucose, etc. via a citric acid cycle, some kinds of acyl CoAderivatives play an important role in biosynthesis of cholesterol andfatty acid as well. CoA is essential as an auxiliary factor (coenzyme)for an enzymatically catalyzing reaction (CoA enzyme) concerning such ametabolism, is a substance which is unable to be substituted with othersand is represented by the following formula.

(In the Formula, ACY is an Acyl Group.)

In CoA enzyme, there are various CoA enzymes depending upon thestructure and the substrate (a compound into which an acyl group is tobe introduced) of an acyl group to be transferred. Up to now, manyattempts have been made for the production of a substance using variouskinds of CoA enzymes and, for example, there are examples for theproduction of antibiotics and drugs, various chemical substancesutilizing a polyketide synthesis route, amino acids, polyhydroxy acids,etc.

In those methods, equimolar acyl CoA is consumed as an acyl groupreceptor. Accordingly, it is required that the necessary acyl CoA isproduced at a low cost.

In any of the above-mentioned methods, acyl CoA in vivo which isfermentationally produced by fermentation or acyl CoA which is producedseparately from the production system is used. When acyl CoA in vivofermentationally produced is used, only a specific acyl CoA produced invivo such as acetyl CoA or malonyl CoA is able to be utilized. In orderto solve such a problem, there has been reported an art where anenzymatic ester exchange is carried out between acetyl CoA and variousfatty acids to produce acyl CoA in vitro. The method where acyl CoAwhich is separately produced from the production system is used has amultiplicity of uses and is a method which has been commonly used butthe acyl CoA which is produced as such is very expensive and it is stillnecessary to use equimolar amount when used for a transfer reaction ofan acyl group.

With regard to a production process of acyl CoA, a chemical syntheticmethod using an acyl chloride, a chemical synthetic method using an acidanhydride, a chemical synthetic method using a mixed acid anhydride withethyl chlorocarbonate, a chemical synthetic method by a thioesterexchange (Z. Naturforsch. 29C, 469-474 (1974); Z. Naturforsch. 30C,352-358 (1975); J. Am. Chem. Soc., 1953, 75, 2520; J. Biol. Chem., 1985,260, 13181) and many other chemical synthetic methods have beengenerally used. However, in many of the chemical synthetic methods,selectivity to thiol group is low in general and there is a problem thatthe yield is lowered by a non-selective acylation reaction. Althoughthese production processes have been used even today, they are merelyused for a laboratory production of acyl CoA.

In order to overcome the weak points of the chemical synthetic method,an enzymatic production process of acyl CoA has been also studiedvigorously. Thus, a method using an acetyl CoA synthetic enzyme, amethod using a fatty acid CoA synthetic enzyme, etc. have been reported(Appl. Microbiol. Biotechnol., 1994, 40, 699-709). However, in thoseenzymatic reaction methods, it is very difficult to obtain the enzymewhich serves as a catalyst in a necessary amount.

With regard to a production process using an enzyme, a study concerninga coupling method in cooperation with a CoA enzyme reaction using it asan acyl CoA reproduction system has been reported. That is, the acyl CoAconsumed by an acyl group transfer reaction is reproduced by anenzymatic reaction and used for the reaction again and there are acoupling method using phosphotransacetylase, a coupling method usingcarnitine acetyltransferase, a coupling method using an acetyl CoAsynthetic enzyme, a coupling method using an α-ketoglutaric aciddehydrogenase, etc. These methods have a high selectivity to thiol groupand particularly an acetyl CoA synthetic enzyme has wide substrateselectivity and is useful because various kinds of acyl CoAs are able tobe generated.

However, these methods reproduce an acyl CoA by enzyme and each of themhas problems including that such an enzymatic reproduction system has aslow reaction rate, enzyme is unstable, ATP and a relatively expensiveauxiliary component are necessary for the reaction and no reaction ispossible in CoA of high concentrations. Thus, unless the price of theaimed product is considerably high, they are generally said to be unableto be industrial production methods in terms of cost if an acyl CoA isused for less than 10,000 times. Consequently, the above-mentionedmethods are not satisfactory as industrial production methods. Althoughthere is an attempt to reproduce an acetyl CoA utilizing a non-enzymaticreaction using an N-acetyl substance of dimethylaminopyridine(Bioorganic Chem., 1990, 18, 131-135), it is a bilayer system using alarge quantity of an organic solvent whereby there is a problem in viewof purification of the product and the method is not suitable for anindustrial production.

As mentioned above, an acyl CoA reproduction system which issatisfactory for enabling the utilization of a CoA enzyme as anindustrial production method has not been known up to now.

Sphingolipid is a lipid derived from a sphingoid base such assphingosine and is present in cell membranes of animals, plants andmicrobes. Although a precise function of human sphingolipid has not beenknown yet, a group of such compounds participates in electric signaltransmittance in a nervous system and stabilization of cell membranes.Sphingoglycolipid has a function in immune system and it has been shownthat a specific sphingoglycolipid functions as a receptor for bacterialtoxin and also probably as a receptor for microbes and viruses.

Ceramide is a specific group of sphingolipid containing sphingosine,dihydrosphingosine or phytosphingosine as a base. Ceramide is a mainlipid component of horny layer which is an upper layer of the skin andhas an important barrier function. It has been known that a topicalapplication of a composition containing a sphingolipid such as ceramideimproves, for example, a barrier function and a moisture-retainingcharacteristic of the skin (Curatolo, Pharm. Res., 4: 271-277 (1987);Kerscher, et al., Eur. J. Dermatol., 1:39-43 (1991)).

It has been known that a sphingoid base per se inhibits the activity ofprotein kinase C which is an important enzyme in a signaling pathway andaccordingly that it mediates several physiological actions. Moreover, asphingoid base is contained in cosmetic compositions or indermatological compositions due to its anti-inflammatory activity andantibacterial activity.

At present, heterosphingolipid preparations for cosmetics are mostlyextracted from animal sources. However, that is a method which isrelatively expensive in an industrial scale and a public concern isincreasing for novel material sources for pure and structurallyspecified sphingolipid which is available from other supplying sourcesthan animal tissues because of, for example, a latency of bovinespongiform encephalopathy (BSE).

It has been found that microbe such as Pichia ciferrii yeast producessphingolipid, sphingosine, phytosphingosine and/or derivatives thereof(Wickerham and Stodola, J. Bacteriol., 80:484-491 (1960)). Such amicrobe provides supplying source for sphingolipid per se and supplyingsource for starting material for production of other commerciallyvaluable compounds and gives an practically applicable substitute to theuse of animal supplying source for those compounds. However, in theproduction by microbes, improvement in productivity is difficult becauseof toxicity of a sphingoid base to microbe cells (Pinto, et al., J.Bacteriol., 174:2565-2574 (1992); Bibel, et al., J. Invest. Dermatol.,93:269-273 (1992)) and there has been a brisk demand for providing moreefficient production process.

In addition, as a result of increasing consciousness to environmentalissues in recent years, there is much more interest in biodegradablemacromolecules being friendly to environment than in syntheticmacromolecules which have occupied the main stream.

Polyhydroxy alkanoate (hereinafter, may be abbreviated as PHA) which isone of biodegradable macromolecules is a polyester being usuallyproduced by a fermentation production of microbes and receiving publicattention due to its high biodegradability and 90 or more kinds havebeen known (FEMS Microbiol. Lett., 1995, 128, 219-228). Among them,research and development have been promoted for poly(3-hydroxybutyrate)(hereinafter, may be abbreviated as PHB), poly(3-hydroxyvalerate)(hereinafter, may be abbreviated as PHV) andpoly(3-hydroxybutyrate-co-3-hydroxyvalerate) (hereinafter, may beabbreviated as PHB-co-PHV) due to ease of produce and goodcharacteristics (Japanese Laid-Open Patent Publication No. 57-150393(U.S. Pat. No. 4,393,167), Japanese Laid-Open Patent Publication No.59-220192 (European Patent Laid-Open No. 0114086), Japanese Laid-OpenPatent Publication No. 63-226291 (European Patent Laid-Open No. 0274151)and Japanese Laid-Open Patent Publication No. 63-269989). However, thereare many problems in PHA that, the productivity is low in the productionby fermentation of microbe in order to accumulate PHA in microbe cellsand that, in addition, it takes much cost for purification by crushingmicrobes and extracting PHA.

Since then, analysis of the mechanism of fermentation production hasproceeded, which increased accumulated concentration of PHA into themicrobe cells significantly, and also analysis of mechanism ofaccumulated state of PHA into microbe cells has proceeded, which loweredcost for extraction and purification of PHA from microbes whereby actualproduction of PHA using microbes has started.

In addition, since it has been in the meanwhile clarified that there arevarieties of microbes which produce PHA, research and development of PHAother than PHB, PHV and PHB-co-PHV has made substantial progress andresearch and development of copolymers for improving the physicalproperty have been also carried out (Japanese Laid-Open PatentPublication Nos. 63-269989, 64-048821, 01-156320, 01-222788 and05-093049).

However, since a production process of PHA by fermentation production ofmicrobe proceeds via a complicated biometabolic path, the desired PHA isnot always produced and, moreover, variation of PHA is limited as well.Further, depending upon a method for controlling the fermentationproduction, a desired homopolymer is not produced but a copolymer isformed and, reversely, a homogeneous copolymer in a desiredpolymerization ratio is not always produced in a production of copolymer(FEMS Microbiol. Rev., 1992, 103, 207-214). In addition, in apurification step, since a desired PHA is taken out from microbe cellscontaining many kinds of compounds, there is a limitation in improvingpurity in an industrial production. As such, production of PHA byfermentation of microbes has various problems.

On the other hand, by a genetic recombination technique which hasquickly progressed in recent years, gene of polyhydroxyalkanoatesynthase (PHAS) which is an enzyme copolymerizing PHA was isolated and,by enhancing its expression, improvement of production of PHA has beenalso attempted (Japanese Laid-Open Patent Publication Nos. 07-265065,10-108682 and 2001-516574 (WO 99/14313)).

Further, it is now also possible to separate and purify PHAS in largequantities using a genetic recombination technique and a method forpolymerization of PHB in vitro without using a microbe fermentation hasbeen developed whereby homogeneous and highly pure PHB is able to beproduced (Proc. Natl. Acad. Sci., 1995, 92, 6279-6283,; Int. Symp.Bacterial Polyhydroxyalkanoates, 1996, 28-35; Eur. J. Biochem., 1994,226, 71-80; Appl. Microbiol. Biotechnol., 1998, 49, 258-266;Macromolecules, 2000, 33, 229-231).

After that, it has been shown that PHA other than PHB is also able to besynthesized by the similar in vitro polymerization method and there isno limitation on variation of PHA which has been unable to be achievedby a microbe fermentation method whereby it is suggested that variationof PHA is significantly expanded (Biomacromolecules, 2000, 1, 433-439;Appl. Microbiol. Biotechnol., 2001, 56, 131-136; Macromolecules, 2001,34, 6889-6894). In that method, it is also possible to synthesizecopolymers in addition to homopolymers.

However, acyl CoA is to be used as a starting substance for thepolymerization in an in vitro polymerization method, but, as mentionedabove, there are various problems for the synthesis of acyl CoA.

Accordingly, there has been a demand for suppressing the amount of anacyl CoA used very small and also for developing a production process ofmacromolecular compounds where other compound which is easilysynthesized industrially is used as a starting substance.

On the other hand, in an in vitro polymerization method, acyl CoA isused as a substrate for enzyme and the enzyme reacts whereupon PHA ispolymerized and, at the same time, liberated CoA is discharged into thereaction system (refer to the following formula).

(In the formula, R⁰ is an organic group wherein R⁰—SH is CoA; R¹ is anyalkylene; and n is an integer corresponding to degree ofpolymerization.)

As such, each time when a reaction of acyl group transfer from acyl CoAtakes place, one repeating unit is added whereupon one molecule of CoAis released.

In an in vitro polymerization method, this CoA remains in a reactionsystem in its free state just to be accumulated therein and the yield ofa macromolecular polymerization reaction does not exceed the equivalentamount of the acyl CoA which is put into the reaction system. Therefore,productivity of PHA is very low and cost of PHA manufactured by an invitro polymerization method is nothing but quite expensive. As thepolymerization proceeds further, CoA concentration in the reactionsystem increases whereby there are concerns about an inhibition effectto the enzymatic reaction as well.

Incidentally, as an effective utilization method of CoA which is presentin a high concentration in the reaction system in a free state, itsreproduction is attempted as well (FEMS Microbiology Letters, 1998, 168,319-324). That is, acetic acid, acetyl CoA synthetase and ATP are madecoexisted in a polymerization enzymatic reaction solution whereby CoAwhich is liberated after the polymerization reaction is converted toacetyl CoA and, in addition, propionyl CoA transferase and3-hydroxybutyrate are also made coexisted to give 3-hydroxybutyrate CoAwhich is a substrate for the polymerization catalyst. In that methodhowever, as many as three kinds of enzymes which are very difficult topurify are used and, further, quite expensive ATP is also necessarywhereby it is very difficult to apply it to an industrial productionprocess.

As such, in an in vitro polymerization method, it is necessary to use anacyl CoA as a reaction substrate and, since acyl CoA is very expensive,it is quite difficult to lower the production cost of PHA when acyl CoAis used as a reaction substrate for an industrial production of PHA.Moreover, in a reproduction of CoA to acyl CoA, many kinds of enzymeswhich are difficult to obtain are necessary and, in addition, anexpensive compound such as ATP is necessary. Further, in a productionprocess of PHA using living organism or, particularly, microbe,variation of PHA is limited and, furthermore, there is a highpossibility that copolymer is polymerized due to metabolism in livingbody whereby it is difficult to produce a desired PHA only. In view ofthe above, there has been a demand for developing a production processwhich enables greater variation of PHA, and can lower the productioncost of PHA by using a compound which is easily able to be synthesizedin its production as a starting substance.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide an industrialacyltransferase reaction method using a CoA enzyme in an acyl CoAreproduction system and, particularly, to provide an acyltransferasereaction method which is useful for the production of a physiologicallyactive substance such as biological material.

Another object of the present invention is to provide an entirely novelsemi-synthetic production process to produce a useful sphingoid base byan enzymatic reaction. Still another object is to provide an economicalmethod where an effective production of sphingoid base is possible usinga coenzyme (CoA) of a catalytic amount.

Further object of the present invention is to provide a process for aneffective production by a coenzyme (CoA) of a catalytic amount in theproduction of a biodegradable macromolecular compound useful for anenzymatic reaction.

The present inventors have extensively studied a reproduction system ofacyl CoA concerning efficiency, speed, cost and selectivity and alsowhether a coupling is possible between the reproducing reaction and anenzymatic reaction. As a result, it has been found that a thioesterexchange reaction which is a chemical synthetic method so far used onlyfor preparation is able to be coupled with an enzymatic reaction systemwhereupon the present invention has been achieved.

The thioester exchange reaction proceeds in a system of neutral toweakly basic region and a substrate specificity of acyl group is verybroad whereby it can be coupled with any CoA enzyme showing a reactivitywithin a region in which a thioester exchange reaction can take place.

Further, the present inventors have applied this coupling method to aserine C-palmitoyl transferase which is a key enzyme in a biosynthesispathway for sphingolipid and succeeded in establishing a productionmethod and the like for the production of a sphingoid base which is animportant physiologically active substance by a decarboxylating transferreaction of fatty acid chain via CoA using a tiophenyl fatty acid andserine.

Still further, in order to develop a production process of PHA in highefficiency, the present inventors have eagerly investigated to find anovel synthetic pathway for various compounds related to PHA from anorganic compound. As a result, they have found that, when a thioesterexchange reaction is combined with an in vitro polymerization method, astarting substance for the reaction can be substituted with an easilysynthesizable thiophenyl ester and a reproduction reaction of an acylCoA which is essential in the polymerization reaction is also able to beconducted in the same reaction solution system whereby consumed amountof acyl CoA can be dramatically reduced together with suppression ofconcentration of CoA. Thus, the present invention has now been achieved.

Accordingly, the present invention relates to the followingacyltransferase reaction method.

-   1. A method for acyltransferase reaction in which an acyl group of    acyl coenzyme A (acyl CoA) is transferred characterized in that the    reaction is carried out by production and/or reproduction of acyl    coenzyme A from coenzyme A in a reaction system by a chemical    thioester exchange reaction with an acyl group donor which is an    acyl ester of a thiol compound.-   2. The method for acyltransferase reaction according to 1 above,    wherein an acyl group donor, acyl group receptor, coenzyme A and    acyltransferase are contained in the reaction system at the same    time, an acyl group of the acyl group donor is transferred to    coenzyme A by a chemical thioester exchange reaction to give an acyl    coenzyme A and an acyl group of the acyl coenzyme A is transferred    to the acyl group receptor.-   3. The method for acyltransferase reaction according to 2 above,    wherein the method is carried out together with production and/or    reproduction of acyl coenzyme A by an acyl group of the acyl group    donor.-   4. The method for acyltransferase reaction according to 2 above,    wherein the thiol compound is aromatic thiol.-   5. The method for acyltransferase reaction according to 4 above,    wherein the aromatic thiol is thiophenol which may optionally    contain a substituent group(s).-   6. The method for acyltransferase reaction according to 2 above,    wherein the acyl group receptor is amino acid and/or a derivative    thereof.-   7. The method for acyltransferase reaction according to 2 above,    wherein the acyl group receptor is serine and/or a derivative    thereof.-   8. The method for acyltransferase reaction according to 1 or 2    above, wherein the acyltransferase is serine C-palmitoyl    transferase.-   9. The method for acyltransferase reaction according to 8 above,    wherein the serine C-palmitoyl transferase is derived from bacteria    of genus Sphingomonas.-   10. The method for acyltransferase reaction according to 1 or 2    above, wherein the acyltransferase is a sphingosine N-acyl    transferase.-   11. The method for acyltransferase reaction according to 2 above,    wherein the acyltransferase is a macromolecular polymerization    enzyme and a macromolecular compound is synthesized in a reaction in    which an acyl group donor, acyl group receptor, coenzyme A and    acyltransferase are contained in the reaction system at the same    time, an acyl group of the acyl group donor is transferred to    coenzyme A by a chemical thioester exchange reaction to give an acyl    coenzyme A and an acyl group of the acyl coenzyme A is transferred    to the acyl group receptor.-   12. The method for acyltransferase reaction according to 11 above,    wherein an acyltransferase reaction is repeated using acyl coenzyme    A or a product by the acyltransferase reaction as an acyl group    receptor whereby the macromolecular compound is produced.-   13. The method for acyltransferase reaction according to 11 above,    wherein the acyl thioester is acyl ester of aromatic thiol.-   14. The method for acyltransferase reaction according to 13 above,    wherein the acyl ester of aromatic thiol is hydroxyalkanoate    thiophenyl ester.-   15. The method for acyltransferase reaction according to 14 above,    wherein the hydroxyalkanoate thiophenyl ester is 3-hydroxyalkanoate    thiophenyl ester.-   16. The method for acyltransferase reaction according to 15 above,    wherein the 3-hydroxyalkanoate thiophenyl ester is 3-hydroxybutyrate    thiophenyl ester.-   17. The method for acyltransferase reaction according to 11 above,    wherein the macromolecular polymerization enzyme is polyhydroxy    alkanoate synthase.-   18. The method for acyltransferase reaction according to 17 above,    wherein the polyhydroxy alkanoate synthase is derived from genus    Ralstonia.-   19. The method for acyltransferase reaction according to 18 above,    wherein the genus Ralstonia is Ralstonia eutropha.-   20. The method for acyltransferase reaction according to 19 above,    wherein Ralstonia eutropha is Ralstonia eutropha ATCC 17699.-   21. A production process of a sphingoid base using the    acyltransferase reaction described in any of 7 to 9 above.-   22. The product on process according to 21 above, wherein the    sphingoid base is 3-ketodihydrosphingosine.-   23. A production process of a ceramide using the acyltransferase    reaction described in 10 above.-   24. In a production process of a macromolecular compound using the    acyltransferase reaction described in any of 11 to 20 above, a    production process of polyester in which the macromolecular compound    is polyester.-   25. The production process of the polyester according to 24 above,    wherein the polyester is polyhydroxy alkanoate.-   26. The production process of the polyester according to 25 above,    wherein the polyhydroxy alkanoate is poly(3-hydroxy alkanoate).-   27. The production process of the polyester according to 26 above,    wherein the poly(3-hydroxy alkanoate) is poly(3-hydroxy butyrate).

BRIEF DESCRIPTION OF DRAWING

FIG. 1 is a scheme which shows a coupling reaction of a CoA enzymereaction with an acyl CoA reproduction system by a thioester exchangeaccording to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, as shown in FIG. 1, an acyl groupdonor, acyl group receptor, CoA and acyltransferase (CoA enzyme) existin one system and an acyl group transfer reaction is able to be carriedout by a coupling reaction where acyl CoA consumed by the progress ofthe reaction is produced and reproduced by a chemical thioester exchangereaction of the acyl group donor with the coenzyme A in the same systemas an enzymatic reaction.

In an embodiment of the present invention, acyl CoA consumed by theprogress of the reaction is produced and reproduced by a chemicalthioester exchange reaction. As a result, an efficient acyl grouptransfer reaction is able to be achieved only by the presence of a smallamount of acyl CoA which is expensive.

In another embodiment of the present invention, acyl CoA or the productby an acyl group transfer reaction is further made into an acyl groupreceptor. As a result, an efficient polymer producing reaction is ableto be achieved by repetition of an acyl group transfer reaction.

The above-mentioned second embodiment (reaction for the production of apolymer) is a part of the above-mentioned first embodiment (a highlyefficient acyl group transfer reaction) and, hereinafter, the firstembodiment (a highly efficient acyl group transfer reaction) and thesecond embodiment (a reaction for the production of polymer) will beillustrated separately for the sake of convenience.

(1) Highly Efficient Acyl Group Transfer Reaction

(1-1) CoA Enzyme

With regard to the CoA enzyme used in this embodiment, there is noparticular limitation so far as it uses an acyl CoA as an auxiliaryfactor (coenzyme). Examples of such an enzyme are transferases belongingto a series of “EC 2.3.1.x” such as acetylglutamic acid synthase (EC2.3.1.1), acetoacetyl CoA thiolase (EC 2.3.1.9) and serine C-palmitoyltransferase (EC 2.3.1.50). It has been made clear already that thoseenzymes are present in many living organisms and they have beenseparated from various living organisms and purified already (EnzymeNomenclature, 178-199, Academic Press, Inc. (1992)). Among those,enzymes having optimum pH in neutral to weakly basic regions arepreferred. Although those CoA enzymes may be pure ones, it is alsopossible to use catalytic microbe cells having an CoA enzymatic activityor a processed product thereof. In that case however, it is desirablethat influence of enzymes other than for the purpose of using acyl CoAas an auxiliary factor are to be avoided by means of use of defectivemutant, inhibition of activity, inactivating treatment, etc.

(1-2) Acyl Group Donor

With regard to an acyl group donor used in the highly efficient acylgroup transfer according to the present invention, there is nolimitation so far as it is acyl ester of a thiol compound (in thepresent application, it may be merely referred to as “acylthioester”) bywhich a thioester exchange reaction with CoA takes place in anon-catalytic manner, but an acyl ester of aromatic thiol is preferred.Examples of the aromatic thiol are thiophenol, methylthiophenol,chlorothiophenol, 2-mercaptothiazole, 2-mercaptoimidazole,2-mercaptotriazole, 2-mercaptobenzothiazole, 2-mercaptobenzimidazole and2-mercaptopyridine. Particularly preferred examples are acyl ester ofthiophenol (which may be merely referred to as “thiophenyl ester” in thepresent application including the case where the phenyl group issubstituted).

With regard to an acyl group corresponding to the acyl ester thiol, anyacyl group may be fundamentally used without limitation. Examplesthereof are a C₂ to C₂₀ saturated or unsaturated aliphatic acyl groupsuch as acetyl (CH₃CO—), propionyl (CH₃CH₂CO—), butyryl (CH₃CH₂CH₂CO—),isobutyryl ((CH₃)₂CHCO—), acryloyl (CH₂═CH—CO—) methacryloyl(CH₂═C(CH₃)—CO—), palmitoyl (CH₃—[CH₂]₁₄—CO—), stearoyl(CH₃—[CH₂]₁₆—CO—) and oleoyl (CH₃—[CH₂]₆—CH═CH—[CH₂]₆—CO—) and anaromatic acyl group such as benozyl. Of course these are mereexemplifications and, for example, an alkyl chain of the aliphatic acylgroup may be substituted and a part of or all of them may be cyclic. Thearomatic ring of the aromatic acyl group may be a carbocyclic ring, ahetero ring or a fused ring and may be optionally substituted. Examplesof the substituent group are a hydroxyl group, an alkyl group, an arylgroup, an aralkyl group, an amino group and halogen such as chlorine andbromine.

(1-3) Acyl Group Receptor

With regard to the acyl group receptor used in the highly efficient acylgroup transfer reaction according to the present invention, there is nolimitation so far as it is able to be used as a substrate for the aboveCoA enzyme. When a substrate specificity of the enzyme is changed by thereaction condition or when a mutant where the substrate specificity ismodified by means of protein engineering is used, a substance which isnot usually an appropriate substrate for the enzyme is also able to beused as an acyl group receptor.

Preferred acyl group receptors are amino acid and amino acid derivativeand particularly preferred ones are natural amino acid and nonnaturalamino acid. For example, when an amino acid is serine and an enzyme isserine C-palmitoyl transferase, an efficient synthetic reaction for3-ketodihydrosphingosine is resulted. When an acyl group receptor issphingosine which is an amino acid derivative and an enzyme issphingosine N-acyl transferase, an efficient synthetic reaction forceramide is resulted. Incidentally, a product in the acyl group transferreaction does not always have a transferred acyl group as it is but maybe decarboxylated or rearranged under the reaction condition and,generally, it depends upon the enzyme and the substrate used therefor.

(1-4) Reaction Condition

The CoA used in the highly efficient acyl group transfer reactionaccording to the present invention may be a product manufactured by anyof the methods of chemical synthesis, semi-synthesis, biologicalfermentation, etc. so far as it is able to function as CoA.

With regard to the reaction system used in the highly efficient acylgroup transfer reaction according to the present invention, there is nolimitation so far as it is a system where an ester exchange reactionbetween an acyl group donor and CoA and an acyl group transfer reactionfrom an acyl CoA to an acyl group receptor by the CoA enzyme usedproceed at the same time and it is possible to use an aqueous uniformsystem, an organic solvent uniform system or a bilayer system of anorganic solvent and water.

With regard to the reaction of the present invention, it may take placeat a temperature where stability of the CoA enzyme is ensured and thereaction proceeds. Usually, the reaction temperature is 10° C. to 45° C.and, preferably, 20° C. to 40° C.

There is no particular limitation on the concentration in the reactionof the present invention so far as stability of the CoA enzyme isensured and the reaction proceeds.

The reaction system may be an open type or a tightly closed type and,when smell or the like is a problem, the reaction may be carried out ina tightly closed system.

(2) Reaction for Production of Macromolecular Compound

(2-1) Macromolecular Compound

As mentioned above, the present invention is also useful as a reactionfor the production of a macromolecular compound and, to be morespecific, it relates to a production process of a macromolecularcompound where a macromolecular compound is synthesized from a thioesterin a solution where a thioester exchange reaction and a macromolecularpolymerization enzyme reaction are made co-existed.

With regard to the macromolecular compound synthesized in the presentinvention, there is no limitation so far as it is a macromolecularcompound synthesized from a thioester in a solution where a thioesterexchange reaction and a macromolecular polymerization enzyme reactionare made co-existed, and an example thereof is a polyhydroxy alkanoate(PHA) which has been reported to be produced mainly by fermentationproduction of microorganism up to now. It has been known that 90 or morekinds thereof are available (FEMS Microbiol. Lett. 1995, 128, 219). Morespecific examples are a compound having C₂ or higher alkyl chain in aside chain, which compound may have C₆ or more or C₁₀ or more long-chainalkyl group; a compound having a branched alkyl group in the side chain;a compound having a phenyl ring in the side chain, which compound mayhave a modified group on the phenyl ring; a compound having a phenoxyring in the side chain, which compound may have a modified group on thephenoxy ring; a compound having a double bond or a triple bond in theside chain, which may exhibit a good polymerizing property; a compoundhaving a halogen element in the side chain; a compound having a cycloring in the side chain; and a compound having an epoxy ring in the sidechain. Such a PHA may be a homopolymer or may be a copolymer comprisingtwo or more kinds of units.

To be more specific, the PHA may include those mentioned in Int. J.Biol. Macromol., 1990, 12, 92-101, etc. for an alkyl group; in Macromol.Chem., 1990, 191, 1957-1965, Macromolecules, 1991, 24, 5256-5260,Macromolecules, 1996, 29, 1762-1766, etc. for a phenyl ring; inMacromolecules, 1996, 29, 3432-3435, Macromol. Chem. Phys., 1994, 195,1665-1672, etc. for a phenoxy ring; in Appl. Environ. Microbiol. 1988,54, 2924-2932, Int. J. Biol. Macromol., 1990, 12, 85-91, J. Polym. Sci.Part A, 1995, 33, 1367-1374, Macromolecules, 1994, 27, 1675-1679,Macromolecules, 1998, 31, 1480-1486, etc. for a double bond; inMacromolecules, 1998, 31, 4760-4763, etc. for a triple bond; inMacromolecules, 1990, 23, 3705-3707, J. Chem. Soc. Polym. Commun., 1990,31, 404-406, Macromolecules, 1992, 25, 1852-1857, Macromolecules, 1996,29, 4572-4581, etc. for a halogen element; and in Macromolecules, 1999,32, 7389-7395, etc. for an epoxy ring; and carbon numbers of the PHA arevarious as well.

Specific examples of the macromolecular compound include poly(3-hydroxyalkanoate) and poly(4-hydroxy alkanoate) which have been well known tobe produced mostly by living body and, particularly, by fermentationproduction of microbe. To be more specific, poly(3-hydroxy butyrate) maybe particularly listed. Needless to say, the above-mentioned ones aremere examples and all macromolecular compounds containing apolymerization unit which is capable of forming a macromolecule by theprocess of the present invention are included. In addition, acombination of plural kinds of polymerization units may be included.There is no particular limitation on the degree of polymerization so faras the enzymatic reaction proceeds.

Although there is no particular limitation on the enzymaticpolymerization reaction which can be used for the present invention, itis possible to list a reaction using, for example, hydroxy alkanoatecoenzyme A as acyl coenzyme A and, in that case, PHA is produced as themacromolecular compound.

(2-2) CoA Enzyme

For the macromolecular polymerization enzyme used in the presentinvention, any enzyme may be used so far as it is a macromolecularpolymerization enzyme which synthesizes a macromolecular compound usingthe substance produced by the thioester exchange reaction of the presentinvention as a substrate. For example, in case hydroxy alkanoate CoA isused as a substrate for the production of PHA, it is possible to usepolyhydroxy alkanoate synthase (PHAS) which is an enzyme. With regard toa method for the preparation of a macromolecular polymerization enzyme,various methods such as a method of extracting and purifying of anenzyme from living organism cells and a method of extracting andpurifying of an enzyme from an incubated product of living organism maybe used, and an example is that PHAS is able to be extracted andpurified from microbe cells. However, in the conventional extracting andpurifying method, the amount of the enzyme to be obtained is limited tobe very small and, therefore, in recent years, gene of PHAS is isolatedutilizing a genetic recombination technique (J. Biol. Chem., 1989, 264,15298-15303; J. Bacteriol., 1988, 170, 4431-4436; J. Bacteriol., 1988,170, 5837-5847) to highly express whereby the macromolecularpolymerization enzyme is able to be separated and purified in a largeamount (J. Biochemistry, 1994, 33, 9311-9320; Protein Expression Purif.,1996, 7; 203-211) In addition to using as it is, the enzyme used in themacromolecular polymerization reaction of the present invention may alsobe used after being modified such as an immobilized enzyme.

Although there is no particular limitation on the source of livingorganisms wherefrom the macromolecular polymerization enzyme is derived,examples thereof are genus Ralstonia, genus Pseudomonas, genusChromatium, genus Ectothiorhodospira and many microbes which have beenwell known for the production of PHA. It is also possible to obtain amacromolecular polymerization enzyme from a genetic recombinant having amacromolecular polymerization enzyme gene derived from those livingorganisms as a donor. An example is that gene of PHAS of Ralstoniaeutropha ATCC 17699 is isolated, its recombinant Escherichia coli isprepared and incubated, and a desired PHAS is extracted and purifiedfrom the incubated product and is able to be used as a catalyst for amacromolecular polymerization reaction.

(2-3) Acyl Group Donor

The acyl group donor used in the macromolecular production reactionaccording to the present invention is the same as that in theabove-mentioned highly efficient acyl group transfer reaction exceptthat the acyl group is able to be a constituting unit for themacromolecular substance and acyl ester of aromatic thiol is preferred.Examples of the aromatic thiol are the same as those mentioned already.

Production process of thioester is mentioned, for example, in J. Am.Chem. Soc., 1973, 22, 5829.

The thioester is able to be subjected to a thioester exchange reactionto easily convert to an acyl CoA which is thioester of CoA byco-existing with CoA salt under an alkaline condition (Int. Symp.Bacterial Polyhydroxyalkanoates, 1996, 28-35).

(2-4) Acyl Group Receptor

In an in vitro polymerization method, CoA thioester is used as asubstrate for the enzyme and PHA is polymerized by the reaction of theenzyme and, at the same time, a liberated CoA is released into thereaction system. The CoA thioester contains both of that which waspoured thereinto at the beginning of the reaction and that which wasproduced as the reaction proceeded and, anyway, the products are apolymer and CoA (cf. the following formula).

(In the formula, R⁰ is an organic group wherein R⁰—SH stands for CoA; R¹is any alkylene; and n is an integer corresponding to degree ofpolymerization.)

In the present invention, the above two reactions, i.e. a thioesterexchange reaction and a macromolecular polymerization reaction arecombined and are carried out by making them to coexist in a reactionsystem to produce a macromolecular compound. Thus, CoA which isliberated into a reaction system after the polymerization reaction andhas not been utilized again is made to react with thioester which is putinto the reaction system whereby thioester of CoA is synthesized againand used as a substrate for the polymerization reaction once again (cf.the following formula).

As a result, it is now possible to obtain a macromolecular compound as aproduct more than the equivalent amount of the CoA put into the reactionsystem. Particularly when the turnover of the reaction where the CoA isrepeatedly thio-esterified and liberated becomes higher, it is possibleto significantly lower the industrial production cost of themacromolecular compound.

A higher CoA concentration in the reaction system becomes high as thepolymerization proceeds may have an adverse effect on the enzymaticreaction, and the present invention is very effective in enhancing theproductivity of PHA by suppressing the concentration. As such, thepresent invention is a process for producing PHA in high efficiency.

There is no particular limitation on the reaction condition of thepresent invention and that is similar to those in the highly efficientacyl group transfer reaction. As an example of the condition to promotethe enzymatic reaction, the reaction temperature is 0° C. to 60° C.,preferably 10° C. to 50° C., more preferably 20° C. to 40° C. Simply, itis also possible to conduct the reaction at room temperature. Thedesirable pH range to conduct the reaction is 3 to 12, preferably 5 to10, more preferably 7 to 9.

As used herein, the state where the thioester exchange reaction and themacromolecular polymerization reaction co-exist is a state where thethioester exchange reaction and the macromolecular polymerizationreaction are present in the same aqueous solution, organic solvent or amixed solution thereof or a state where they are present in the samereaction container in a solution or in plural solutions under a mixedstate or a separated state. The separated state of the plural solutionsin layers includes a state where a layer is present in oil drops or in avisibly suspended state. Anyway, it is sufficient if the state necessaryfor the thioester exchange reaction and the macromolecularpolymerization reaction is maintained in a united manner. As a startingsubstance therefor, a thioester which is able to be efficiently producedindustrially is to be made into thioester of CoA in the reaction systemand used as a substrate for the polymerization reaction to produce amacromolecular compound.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will now be illustrated in more detail hereinafteralthough the present invention is not limited by those Examples at all.

EXAMPLE 1 Synthesis of Acyl Thiophenol (Thiophenyl Palmitate)

Anhydrous dichloromethane (6 mL) was added to a well-dried andnitrogen-substituted flask and well stirred with cooling on ice. A 2Mtrimethyl aluminum (2 mL) was slowly added thereto. Further, thiophenolwas slowly added thereto. After the mixture was stirred at roomtemperature for 1 hour and 30 minutes, ethyl palmitate dissolved in 6 mLof anhydrous dichloromethane was slowly added thereto to conduct areaction. The reaction was monitored by TLC. After completion of thereaction, 20 mL of dichloromethane was added to the reaction solutionand then a 3% aqueous solution of hydrochloric acid was added until nomore generation of bubbles was observed. The solution was transferred toa separating funnel, washed with a 3% aqueous solution of hydrochloricacid for three times and with saturated saline for two times, dried overmagnesium sulfate, filtered to remove magnesium sulfate and the filtratewas concentrated using an evaporator to give an oily solution in darkyellow color. This was separated and purified by a silica gel columnchromatography (eluent:hexane/ethyl acetate=2/1) to give thiophenylpalmitate.

EXAMPLE 2 Preparation of Serine C-Palmitoyl Transferase (SPT)

A crude SPT enzyme extract was prepared from a transformant where theabove-mentioned enzyme derived from genus Sphingomonas was cloned toEscherichia coli. Preparation of the Present Transformant and Method forpurification of SPT were in accordance with the descriptions in TheJournal of Biological Chemistry, 276, 18249-18256 (2001) by H. Ikushiro,et al.

Firstly, primers (SEQ ID NO: 1 and SEQ ID NO: 2) coding N-terminalsequence and C-terminal sequence were prepared from total base sequenceof the above SPT and a DNA fragment corresponding to the SPT codingregion was made by a PCR under the following condition using achromosome DNA of Sphingomonas paucimobilis as a template. At that time,NcoI site for connecting to a vector and Hind III site were formed forthe primer for N-terminal and for the primer for C-terminal,respectively.

[Primers] Primer for N-terminal: 5′-accatgaccgaagccgccgctca-3′ (SEQ IDNO: 1) Primer for C-terminal: 5′-taagctttcagccgatgacgccg-3′ (SEQ ID NO:2)[Composition of Reaction Solution]

LA Taq polymerase

Standard buffer attached to said enzyme

Template chromosome <1 μg

Primer: each 1 μM

dNTP: each 200 μM

Liquid volume: 25 μL to 100 μL

[Reaction Condition]

Denaturation temperature: 94° C. for 30 seconds

Annealing temperature: 40+0.25° C./cycle for 30 seconds

Elongation temperature: 72° C. for 90 seconds

The prepared PCR fragment was subjected to an agarose gelelectrophoresis, extracted from the gel and recovered by means of acolumn. The fragment was treated with a restriction enzyme NcoI-HindIIIand ligated with NcoI-HindIII fragment of a plasmid pET21d and a BL21(DE3) strain of the host Escherichia coli was transformed thereby.

The resulting transformant was incubated on 5 mL of an LB mediumcontaining 50 ppm of ampicillin at 35° C. for 16 hours and cells wererecovered by centrifugation and washed with physiological saline. Thewashed cells were re-suspended in 2 mL of buffer for SPT (20 mM ofphosphate buffer (pH 6.5, containing 0.1 mM of EDTA, 5 mM of DTT, 0.1 mMof AEBSF (protease inhibitor) and 0.02 mM of PLP)) and crushed for about10 minutes using an ultrasonic crusher with ice-cooling and anon-crushed product was removed by centrifugation (12,000 rpm for 10minutes) to give a crude enzyme extract.

EXAMPLE 3 Coupling of Ester Exchange Reaction with CoA EnzymaticReaction (Uniform Aqueous System)

CoA sodium salt (2 mg) and 1 mg of L-serine were dissolved in 5 mL of100 nM HEPES-NaOH buffer (containing 10 μM PLP, pH 8.0) and well stirredusing a magnetic stirrer. That was mixed with a solution of 3.5 mg ofthiophenyl palmitate in 0.1 mL of acetonitrile. While the stirring speedwas made reduced to such an extent that a gentle mixing was resulted,0.5 mL of crude SPT enzyme solution was added and the mixture was madeto react at 37° C. for 24 hours. The solution was made alkaline with 1mL of 2N ammonia solution and the product in the solution was extractedand recovered with 5 mL of chloroform/methanol (2:1 (v/v)). The extractliquid was filtered, appropriately concentrated and subjected to aquantitative determination for 3-ketodihydrosphingosine according to thefollowing analytic method whereupon the produced amount was about 1.0mg.

[Analysis of Sphingosines]

Quantitative analysis of sphingosines was carried out using a TLC-FID(Iatroscan) according to a method mentioned in Journal ofChromatography, 284, 433-440 (1984) by M. Tanaka, et al. That is, 1 μLof a solution where 1 to 10 mg of dihydrosphingosine (sphinganine) or3-ketodihydrosphingosine as a standard sample was dissolved in 1 mL ofmethanol was subjected to a chromato-rod S II (silica gel) and developedby a primary developing solution (chloroform-methanol-15N ammoniasolution=60:10:1). The rod after the development was subjected to anIatroscan TH-10 TLC/FID Analyser (manufactured by Iatron) whereuponsphingosines were detected and quantified.

With regard to more detailed quantitative analysis of sphingoid bases,the product was made into a fluoroderivative and then separated andanalyzed by a high-performance liquid chromatography by referring, forexample, to a literature (Analytical Biochemistry, 298 (2001), 283-292).

The reaction solution (75 μL) to be described later was taken and 425 μLof 70.6 mM triethylamine/ethanol solution was added thereto and stirredtherewith. The precipitate was removed by centrifuging for 5 minutes,100 μL of the supernatant liquid was taken in an HPLC sample vial (300μL small amount insert), 20 μL of an AQC reagent (manufactured byWaters) solution was added thereto and the mixture was stirredimmediately. After being made to react at room temperature for 40minutes or longer, the product was analyzed according to the followingHPLC condition.

Main device: LC-VP series (Shimadzu Corporation) (pump: LC-10 ADVP;column oven: CTO-10ACVP; auto-sampler: SIL-10AF; system controller:SCL-10AVP)

Detector: Fluorescence detector 821-FP (Nippon Bunko), Ex. 244 nm, Em.398 nm, Gain×100

Column: Shodex F-511A, 35° C.

Eluent: Acetonitrile/methanol/water/trimethylamine=480/320/190/7; 1.5ml/min.

Method for regeneration of column: In the regeneration method of theliterature, column pressure is apt to be too high, which is likely tocause error, so it was modified as follows.

Analytical cycle: A column regenerating system is placed for each sampleanalysis. Upon regeneration, no sample was infused.

COMPARATIVE EXAMPLE 1 CoA Enzymatic Reaction without Using a CouplingSystem

Palmitoyl CoA (10 mg) and 1 mg of L-serine were dissolved in 5 mL of 100mM HEPES-NaOH buffer (containing 10 μM PLP; pH 8.0) and well stirredusing a magnetic stirrer. While the stirring speed was reduced to suchan extent that a mild mixing was resulted, 0.5 mL of a crude SPT enzymesolution was added and the mixture was made to react at 37° C. for 24hours. The solution was made alkaline with 1 mL of 2N ammonia solutionand the product in the solution was extracted and recovered with 5 mL ofchloroform/methanol (2:1, (v/v)). The extract liquid was filtered,concentrated appropriately and analyzed by the same manner as in Example3 whereupon the produced amount of 3-ketodihydrosphingosine was about0.02 mg.

EXAMPLE 4 Coupling of the Ester Exchange Reaction with the CoA EnzymaticReaction (Oil-Water Bilayer System)

CoA sodium salt (2 mg) and 1 mg of L-serine were dissolved in 5 mL of100 mM HEPES-NaOH buffer (containing 10 μM PLP; pH 8.0) and well stirredusing a magnetic stirrer. A solution prepared by dissolving 3.5 mg ofthiophenyl palmitate in 5 mL of hexane was mixed therewith. While thestirring speed was reduced to such an extent that a mild mixing wasresulted, 0.5 mL of a crude SPT enzyme solution was added and themixture was made to react at 37° C. for 24 hours. The solution was madealkaline with 1 mL of 2N ammonia solution and the product in thesolution was extracted and recovered with 5 mL of chloroform/methanol(2:1, (v/v)). The extract was filtered, concentrated appropriately and3-ketodihydrosphingosine was quantified by the same manner as in Example3 whereupon the produced amount was about 2.2 mg.

Hereinafter, examples of the process for macromolecularization accordingto the present invention will be listed.

REFERENTIAL EXAMPLE 1 (1) Synthesis of 3-Oxobutyrate Ethyl Ester

On ice, 3.9 g of Meldrum's acid was dissolved in 18 ml of dehydrateddichloromethane in a dry flask and stirred and a solution of 4.3 g ofpyridine and 2.2 g of acetyl chloride dissolved in 18 ml of dehydrateddichloromethane was slowly added thereto under a nitrogen stream. Themixture was stirred at 0° C. for one hour and then at room temperaturefor two hours. The mixed solution was transferred to a separatingfunnel, washed with 3% hydrochloric acid solution for two times and withsaturated saline for two times, dried over magnesium sulfate andevaporated in vacuo to give 3.6 g of crude oily acylated Meldrum's acidin dark orange color which solidified slowly. The crude acyl Meldrum'sacid was refluxed in 80 ml of dehydrated ethanol. At that time,generation of carbon dioxide was observed. The solvent was removed byevaporation to give 1.3 g of crude 3-oxobutyrate ethyl ester as a redoil. This was purified by a column chromatography (20 cm×1 cm diameter;eluent was hexane:ethyl acetate=2:1) using silica gel 60 to give 0.60 gof pure 3-oxobutyrate ethyl ester as a slightly yellow oil. The yield tothe unpurified substance was 46%. Result of NMR analysis of thiscompound is as follows.

¹H NMR (in CDCl₃) δ4.20 (q, J=7.1 Hz, 2H), 3.47 (s, 2H), 2.27 (s, 3H),1.28 (t, J=7.1 Hz, 3H);

¹³C NMR (in CDCl₃) δ201.06, 167.44, 61.52, 50.29, 30.32, 14.29

REFERENTIAL EXAMPLE 1 (2) Synthesis of 3-Hydroxybutyrate Ethyl Ester

A solution of 75.6 mg of sodium borohydride dissolved in 2 ml ofdehydrated ethanol was stirred in a dry flask and a solution of 520 mgof 3-oxobutyrate ethyl ester in 2 ml of dehydrated ethanol was slowlyadded thereto. The mixture was stirred at room temperature for two hoursand then 4 ml of water was added. The mixed solution was transferred toa separating funnel, extracted with dichloromethane for two times, driedover magnesium sulfate and evaporated in vacuo to give 282 mg of3-hydroxybutyrate as a light yellow oil. Result of NMR analysis of thiscompound is as follows.

¹H NMR (in CDCl₃) δ 4.17 (q, J=7.1 Hz, 2H), 4.17 (m, 1H), 2.46 (m, 2H),1.28 (t, J=7.1 Hz, 3H), 1.23 (d, J=6.3 Hz, 3H);

¹³C NMR (in CDCl₃) δ 172.93, 64.28, 60.68, 42.91, 22.49, 14.18

REFERENTIAL EXAMPLE 1 (3) Synthesis of 3-Hydroxybutyrate ThiophenylEster

In a dry flask on ice, 6 ml of dehydrated dichloromethane was stirredand 2 ml of 2M trimethyl aluminum was slowly added thereto under anitrogen stream. After that, 2 mmol of thiophenol was slowly addedthereto. The mixture was stirred at room temperature for 30 minutes andthen 3-hydroxybutyrate dissolved in 6 ml of dehydrated dichloromethanewas added thereto. The reaction was monitored by a TLC. To this mixedsolution was added 20 ml of dichloromethane and, until generation ofbubbles stopped, 20 ml of 3% hydrochloric acid solution was added. Themixed solution was transferred to a separating funnel, washed with 3%hydrochloric acid solution for two times and with saturated saline fortwo times, dried over magnesium sulfate and evaporated in vacuo to give532 mg of crude oily 3-hydroxybutyrate thiophenyl ester in dark yellowcolor. This was purified by a column chromatography (20 cm×1 cmdiameter; eluent was hexane:ethyl acetate=2:1) using silica gel 60 togive 125 mg of pure 3-hydroxybutyrate thiophenyl ester as a transparentoil. The yield to the unpurified substance was 24%. Result of NMRanalysis of this compound is as follows.

¹H NMR (in CDCl₃) δ 7.38 (s, 5H), 4.33 (m, 1H), 2.83 (m, 2H), 1.25 (d,3H);

¹³C NMR (in CDCl₃) δ198.24, 134.90, 130.07, 129.69, 127.61, 65.23,52.02, 22.85

REFERENTIAL EXAMPLE 1 (4) Synthesis of 3-Hydroxybutyrate CoA Thioester

A solution of 9.8 mg of 3-hydroxybutyrate thiophenyl ester dissolved in0.1 ml of acetonitrile was added to a solution of 39.5 mg of coenzyme Asodium salt dissolved with stirring in 0.5 ml of 100 mM calciumphosphate buffer (pH 8.0) in a small glass bottle. The mixture wasstirred at room temperature for three hours and then 0.13 ml of 1Mphosphoric acid was added thereto. The mixed solution was washed with0.5 ml of diethyl ether for three times and evaporated in vacuo to give30 mM of 3-hydroxybutyrate CoA thioester solution.

REFERENTIAL EXAMPLE 2 (1) Synthesis of (R)-3-Hydroxybutyrate ThiophenylEster

tert-Butyl dimethylsilyl chloride (2.53 g) was dissolved in anhydrousdimethylformamide and stirred, 3.4 g of imidazole was added thereto andthe mixture was stirred for 15 minutes on ice in an nitrogen stream.Then 0.5 g of (R)-3-hydroxybutyrate dissolved in anhydrousdimethylformamide was further added thereto followed by stirring at roomtemperature for one night. To the reaction solution was added 60 ml ofsaturated saline and extraction with a solution of diethylether:petroleum ether=1:3 was repeated for five times. The extractliquid was dried over magnesium sulfate and evaporated in vacuo. Thiswas dissolved in a solution of methanol:tetrahydrofuran=2:1, 10 ml of anaqueous solution containing 1.5 g of potassium carbonate was added andthe mixture was stirred at room temperature for one night. The reactionsolution was diluted with saturated saline, adjusted to pH 3.0 with 1Msulfuric acid and extracted with a solution of diethyl ether:petroleumether=1:3 for five times. The extract liquid was dried over magnesiumsulfate, evaporated in vacuo and dried in vacuo to give3-(tert-butyldimethylsilyl) butyrate. On ice, 870 mg of3-(tert-butyldimethylsilyl) butyrate and 452 mg of thiophenol weredissolved in 6 ml of dichloromethane, a solution of 846 mg ofdicyclohexyl carbodiimide dissolved in 2 ml of dichloromethane was addedthereto followed by stirring and the mixture was stirred at roomtemperature for ten hours. Diethyl ether (20 ml) was added thereto, themixture was filtered, the solvent was removed by evaporation and a flushchromatography was conducted (eluent was hexane containing 5% of ethylacetate) to give 330 mg of 3-(tert-butyldimethylsilyl) butyratethiophenyl ester. This was dissolved in 2 ml of acetonitrile and then anacetonitrile solution containing 6 ml of 5% hydrogen fluoride wasfurther added thereto. After the reaction for 20 minutes, a saturatedsodium hydrogen carbonate solution was added until no more bubble wasgenerated, the mixture was extracted with diethyl ether and the extractwas washed with saturated saline, dried over magnesium sulfate andevaporated in vacuo to give 81 mg of (R)-3-hydroxybutyrate thiophenylester.

REFERENTIAL EXAMPLE 2 (2) Synthesis of (R)-3-Hydroxybutyrate CoAThioester

A 3-hydroxybutyrate CoA thioester solution was prepared by the samemanner as in the synthesis of 3-hydroxybutyrate CoA thioester.

REFERENTIAL EXAMPLE 3 (1) Synthesis of 3-Oxovalerate Ethyl Ester

On ice, 3.9 g of Meldrum's acid was dissolved in 18 ml of dehydrateddichloromethane in a dry flask and stirred and a solution of 4.3 g ofpyridine and 2.5 g of propionyl chloride dissolved in 18 ml ofdehydrated dichloromethane was slowly added thereto under a nitrogenstream. The mixture was stirred at 0° C. for one hour and then at roomtemperature for two hours. The mixed solution was transferred to aseparating funnel, washed with 3% hydrochloric acid solution for twotimes and with saturated saline for two times, dried over magnesiumsulfate and evaporated in vacuo to give 3.4 g of crude oily acylatedMeldrum's acid in dark orange color which solidified slowly. The crudeacyl Meldrum's acid was refluxed in 80 ml of dehydrated ethanol. At thattime, generation of carbon dioxide was observed. The solvent was removedby evaporation to give 1.7 g of crude 3-oxovalarate ethyl ester in redoil. This was purified by a column chromatography (20 cm×1 cm diameter;eluent was hexane:ethyl acetate=2:1) using silica gel 60 to give 0.50 gof pure 3-oxovalerate ethyl ester as a slightly yellow oil. The yield tothe unpurified substance was 29%. Result of NMR analysis of thiscompound is as follows.

¹H NMR (in CDCl₃) δ 4.19 (q, J=7.1 Hz, 2H), 3.40 (s, 2H), 2.58 (q, J=7.2Hz, 2H), 1.28 (t, J=7.2 Hz, 3H), 1.08 (t, J=7.2 Hz, 3H);

¹³C NMR (in CDCl₃) δ203.48, 180.07, 61.33, 49.03, 36.32, 14.13, 7.56

REFERENTIAL EXAMPLE 3 (2) Synthesis of 3-Hydroxyvalerate Ethyl Ester

A solution of 75.6 mg of sodium borohydride dissolved in 1 ml ofdehydrated ethanol was stirred in a dry flask and a solution of 288 mgof 3-oxovalerate ethyl ester in 1 ml of dehydrated ethanol was slowlyadded thereto. The mixture was stirred at room temperature for two hoursand then 2 ml of water was added. The mixed solution was transferred toa separating funnel, extracted with dichloromethane for two times, driedover magnesium sulfate and evaporated in vacuo to give 212 mg of3-hydroxyvalerate as a light yellow oil. Result of NMR analysis of thiscompound is as follows.

¹H NMR (in CDCl₃) δ 4.17 (q, J=7.1 Hz, 2H), 3.94 (m, 1H), 2.45 (m, 2H),1.57 (m, 2H), 1.27 (t, J=7.1 Hz, 3H), 0.96 (t, J=7.3 Hz, 3H);

¹³C NMR (in CDCl₃) δ173.30, 69.64, 60.91, 41.46, 29.77, 14.40, 10.07

REFERENTIAL EXAMPLE 3 (3) Synthesis of 3-Hydroxyvalerate ThiophenylEster

In a dry flask on ice, 3 ml of dehydrated dichloromethane was stirredand 1 ml of 2M trimethyl aluminum was slowly added thereto under anitrogen stream. After that, 1 mmol of thiophenol was slowly addedthereto. The mixture was stirred at room temperature for 30 minutes andthen 3-hydroxyvalerate dissolved in 3 ml of dehydrated dichloromethanewas added thereto. The reaction was monitored by a TLC. To this mixedsolution was added 10 ml of dichloromethane and, until generation ofbubbles stopped, 10 ml of 3% hydrochloric acid solution was added. Themixed solution was transferred to a separating funnel, washed with 3%hydrochloric acid solution for two times and with saturated saline fortwo times, dried over magnesium sulfate and evaporated in vacuo to give258 mg of crude oily 3-hydroxyvalerate thiophenyl ester in dark yellowcolor. This was purified by a column chromatography (20 cm×1 cmdiameter; eluent was hexane:ethyl acetate=2:1) using silica gel 60 togive 44 mg of pure 3-hydroxyvalerate thiophenyl ester as a transparentoil. The yield to the unpurified substance was 17%. Result of NMRanalysis of this compound is as follows.

¹H NMR (in CDCl₃) δ 7.39 (s, 5H), 4.04 (m, 1H), 2.82 (m, 2H), 1.60 (m,2H), 0.98 (t, J=7.1 Hz, 3H);

¹³C NMR (in CDCl₃) δ198.30, 134.67, 129.83, 129.46, 127.31, 70.04,50.03, 29.67, 9.96

REFERENTIAL EXAMPLE 3 (4) Synthesis of 3-Hydroxyvalerate CoA Thioester

A solution of 42 mg of 3-hydroxyvalerate thiophenyl ester dissolved in 1ml of acetonitrile was added to a solution of 79 mg of coenzyme A sodiumsalt dissolved with stirring in 2 ml of 100 mM calcium phosphate buffer(pH 8.0) in a small glass bottle. The mixture was stirred at roomtemperature for three hours and then 0.53 ml of 1M phosphoric acid wasadded thereto. The mixed solution was washed with 2 ml of diethyl etherfor three times and evaporated in vacuo to give 33 mM of3-hydroxyvalerate CoA thioester solution.

REFERENTIAL EXAMPLE 4 Preparation and Purification of Enzyme

A restriction enzyme EcoRI and SmaI fragment (about 5 kbp) were digestedfrom a genomic DNA of Ralstonia eutropha ATCC 17699 and cloned to pUC18to give a plasmid pTI 305 containing a PHA synthetic enzyme gene (PHAS).After that, three kinds—fragment of SmaI and BamHI of vector pQE 30(manufactured by Qiagen) and BamHI.NotI fragment (140 bp) of DNAamplified by a PCR using the following two kinds of primers—were mixedand ligated using NotI.StuI fragment (1.6 kbp) of pTI 305 and pTI 305 astemplates whereupon a plasmid PQEREC was prepared. This was introducedinto Escherichia coli BL 21 (pREP4) to prepare Escherichia coli BL21(pQEREC) for the preparation of enzyme. The Escherichia coli wasincubated in 1,000 ml of LB medium at 30° C. for 16 hours so that enzymewas accumulated in cells and the cells were broken by an ultrasonictreatment whereupon a soluble protein in the cells was recovered. Theprotein was passed through an Ni-NTA agarose gel column so that(His)-PhaC (six histidine being added to N-terminal) was specificallyadsorbed with the column. After washing the column, (His)-PhaC waseluted using imidazole and, after dialysis, 10 mg thereof was obtainedas a pure enzyme. Molecular weight of the enzyme by an SDS-PAGE was 65kDa.

Condition for the PCR

Sense primer: aaggatccatggcgaccggcaaaggcgcgg (SEQ ID NO: 3) Antisenseprimer: tgcagcggaccggtggcctcggcctgccc (SEQ ID NO: 4)

Cycle: 30 cycles×(94° C. for 45 seconds, 58° C. for 30 seconds and 72°C. for 60 seconds)

EXAMPLE 5 Polymerization of Poly((R)-3-Hydroxybutyrate)

To 5 ml of 100 mM potassium phosphate solution was added 0.015 mg of theenzyme followed by well stirring at room temperature. While the stirringspeed was reduced to such an extent that a gentle mixing was resultedand temperature of the solution was kept at 30° C., 5 ml of 1 mM CoAsodium solution and 0.5 ml of 20 mM 3-hydroxybutyrate thiophenyl estersolution (being dissolved in a 1:1 solution of 100 mM potassiumphosphate solution and acetonitrile) were added thereto little by littleand the mixture was further made to react at 30° C. for 24 hours. Afterthat, the solution was washed with 20 ml of hexane for three times andthen the product in the solution was extracted and recovered with 10 mlof chloroform. That was repeated for three times. The extract liquid wasfiltered, dropped into 300 ml of methanol and allowed to stand for 24hours. The resulting precipitate was recovered by filtration and driedin a vacuum drier to give 0.4 mg of poly((R)-3-hydroxybutyrate). Itsmolecular weight (GPC calculated as polystyrene) was Mw=970,000. Resultof NMR analysis of the compound is as follows.

¹H NMR (in CDCl₃) δ5.26 (m, H), 2.53 (m, 2H), 1.25 (s, 3H);

¹³C NMR (in CDCl₃) δ169.53, 67.99, 41.16, 20.15

COMPARATIVE EXAMPLE 2 Polymerization of Poly((R)-3-Hydroxybutyrate)

An enzyme (0.015 mg) was added to 5 ml of 100 mM potassium phosphatesolution and well stirred at room temperature. While the stirring speedwas reduced to such an extent that a gentle mixing was resulted andtemperature of the solution was kept at 30° C., 5 ml of 1 mM CoA sodiumsolution was added thereto little by little and the mixture was furthermade to react at 30° C. for 24 hours. After that, the solution waswashed with 20 ml of hexane for three times and the product in thesolution was extracted and recovered with 10 ml of chloroform. That wasrepeated for three times. The extract was filtered, dropped into 300 mlof methanol and allowed to stand for 24 hours. However, no precipitatewas obtained.

EXAMPLE 6 Polymerization of ((R)-3-Hydroxybutyrate)

An enzyme (0.015 mg) was added to 5 ml of 100 mM potassium phosphatesolution and well stirred at room temperature. While the stirring speedwas reduced to such an extent that a gentle mixing was resulted andtemperature of the solution was kept at 30° C., 5 ml of 1 mM3-hydroxybutyrate CoA solution and 0.5 ml of 20 mM 3-hydroxybutyratethiophenyl ester solution (being dissolved in a 1:1 solution of 100 mMpotassium phosphate solution and acetonitrile) were added thereto littleby little and the mixture was further made to react at 30° C. for 24hours. After that, the solution was washed with 20 ml of hexane forthree times and the product in the solution was extracted and recoveredwith 10 ml of chloroform. That was repeated for three times. The extractliquid was filtered, dropped into 300 ml of methanol and allowed tostand for 24 hours. The resulting precipitate was recovered byfiltration and dried by a vacuum drier to give 0.3 mg ofpoly((R)-3-hydroxybutyrate).

COMPARATIVE EXAMPLE 3 Polymerization of Poly((R)-3-Hydroxybutyrate)

An enzyme (0.015 mg) was added to 5 ml of 100 mM potassium phosphatesolution and well stirred at room temperature. While the stirring speedwas reduced to such an extent that a gentle mixing was resulted andtemperature of the solution was kept at 30° C., 5 ml of 1 mM3-hydroxybutyrate CoA was added thereto little by little and the mixturewas further made to react at 30° C. for 24 hours. After that, thesolution was washed with 20 ml of hexane for three times and the productin the solution was extracted and recovered with 10 ml of chloroform.That was repeated for three times. The extract liquid was filtered,dropped into 300 ml of methanol and allowed to stand for 24 hours. Theresulting precipitate was recovered by filtration and dried by a vacuumdrier to give 0.2 mg of poly ((R)-3-hydroxybutyrate).

COMPARATIVE EXAMPLE 4

An enzyme (0.015 mg) was added to 5 ml of 100 mM potassium phosphatesolution and well stirred at room temperature. While the stirring speedwas reduced to such an extent that a gentle mixing was resulted andtemperature of the solution was kept at 30° C., 0.5 ml of 20 mM3-hydroxybutyrate thiophenyl ester solution (being dissolved in a 1:1solution of 100 mM potassium phosphate solution and acetonitrile) wereadded thereto little by little and the mixture was further made to reactat 30° C. for 24 hours. After that, the solution was washed with 20 mlof hexane for three times and the product in the solution was extractedand recovered with 10 ml of chloroform. That was repeated for threetimes. The extract liquid was filtered, dropped into 300 ml of methanoland allowed to stand for 24 hours. However, no precipitate was formed.

EXAMPLE 7 Polymerization of Poly(3-Hydroxyvalerate)

To 5 ml of 100 mM potassium phosphate solution was added 0.015 mg of theenzyme followed by well stirring at room temperature. While the stirringspeed was reduced to such an extent that a gentle mixing was resulted, 5ml of 1 mM (R,S)-3-hydroxyvalerate CoA and 0.5 ml of 20 mM3-hydroxyvalerate thiophenyl ester solution (being dissolved in a 1:1solution of 100 mM potassium phosphate solution and acetonitrile) wereadded thereto little by little and the mixture was further made to reactat room temperature for 24 hours. After that, the solution was washedwith 20 ml of hexane for three times and then the product in thesolution was extracted and recovered with 10 ml of chloroform. That wasrepeated for three times. The extract liquid was filtered, dropped into300 ml of methanol and allowed to stand for 24 hours. The resultingprecipitate was recovered by filtration and dried in a vacuum drier togive 0.3 mg of poly((R)-3-hydroxyvalerate) Result of NMR analysis of thecompound is as follows.

¹H NMR (in CDCl₃) δ 5.12 (m, H), 2.56 (m, 2H), 1.53 (m, 2H), 0.81 (t,3H);

¹³C NMR (in CDCl₃) δ169.71, 72.26, 39.17, 27.23, 9.76

COMPARATIVE EXAMPLE 5 Polymerization of Poly((3-Hydroxyvalerate)

An enzyme (0.015 mg) was added to 5 ml of 100 mM potassium phosphatesolution and well stirred at room temperature. While the stirring speedwas reduced to such an extent that a gentle mixing was resulted, 5 ml of1 mM (R,S)-3-hydroxyvalerate CoA was added thereto little by little andthe mixture was further made to react at room temperature for 24 hours.After that, the solution was washed with 20 ml of hexane for three timesand the product in the solution was extracted and recovered with 10 mlof chloroform. That was repeated for three times. The extract liquid wasfiltered, dropped into 300 ml of methanol and allowed to stand for 24hours. The resulting precipitate was recovered by filtration and driedby a vacuum drier to give 0.1 mg of poly((R)-3-hydroxyvalerate).

EXAMPLE 8 Polymerization of Poly((R)-3-Hydroxybutyrate) from(R)-3-Hydroxybutyrate Thiophenyl Ester

An enzyme (0.015 mg) was added to 5 ml of 100 mM sodium phosphatesolution (pH 7.5) and 1 mM CoA sodium solution and stirred keeping thetemperature of the solution at 30° C. A hexane solution (5 ml) of 10 mM(R)-3-hydroxybutyrate thiophenyl ester was layered thereon. The stirringspeed was reduced to such an extent that a gentle mixing was resultedand reaction was conducted at 30° C. for 24 hours. After the reaction,the hexane layer was removed and the product in the solution wasextracted with 5 ml of chloroform from the aqueous layer. That wasrepeated for two times. The extract liquid was filtered, dropped into200 ml of methanol and allowed to stand at 4° C. for 24 hours. Theresulting precipitate was recovered by filtration and dried by a vacuumdrier to give 1.5 mg of poly((R)-3-hydroxybutyrate). Molecular weight(GPC calculated as polystyrene) was Mw=1,070,000. Reaction turnover ofthio-esterification and liberation of the added CoA was 3.4 times.

COMPARATIVE EXAMPLE 6 Polymerization of Poly((R)-3-Hydroxybutyrate) from(R)-3-Hydroxybutyrate CoA Thioester

An enzyme (0.015 mg) was added to 5 ml of 100 mM sodium phosphatesolution (pH 7.5) and 1 mM (R)-3-hydroxybutyrate CoA thioester solutionand stirred keeping the temperature of the solution at 30° C. Thestirring speed was reduced to such an extent that a gentle mixing wasachieved and reaction was conducted at 30° C. for 24 hours. After thereaction, the hexane layer was removed and the product in the solutionwas extracted with 5 ml chloroform from the aqueous layer. That wasrepeated for two times. The extract liquid was filtered, dropped into200 ml of methanol and allowed to stand at 4° C. for 24 hours. Theresulting precipitate was recovered by filtration and dried by a vacuumdrier to give 0.4 mg of poly ((R)-3-hydroxybutyrate).

COMPARATIVE EXAMPLE 7 Polymerization of Poly((R)-3-Hydroxybutyrate) from(R)-3-Hydroxybutyrate Thiophenyl Ester

An enzyme (0.015 mg) was added to 5 ml of 100 mM sodium phosphatesolution (pH 7.5) and stirred keeping the temperature of the solution at30° C. A hexane solution (5 ml) of 10 mM (R)-3-hydroxybutyratethiophenyl ester was layered thereon. The stirring speed was reduced tosuch an extent that a gentle mixing was resulted and reaction wasconducted at 30° C. for 24 hours. After the reaction, the hexane layerwas removed and the product in the solution was extracted with 5 ml ofchloroform from the aqueous layer. That was repeated for two times. Theextract liquid was filtered, dropped into 200 ml of methanol and allowedto stand at 4° C. for 24 hours. No precipitate was observed.

INDUSTRIAL APPLICABILITY

In accordance with the process of the present invention using anacyltransferase, it is now possible to continuously carry out thereaction and to improve its productivity significantly withoutsupplementary addition of very expensive acyl coenzyme A (acyl CoA).Accordingly, various kinds of compounds are able to be produced by anovel coupling method, which enables utilization of acyltransferase toan industrial production method. According to the present invention, ina production process of sphingoid bases, a thioester exchange reactionis combined with an enzymatic reaction whereby sphingoid bases whichhave been difficult to produce in the conventional fermentation processare able to be accumulated and produced without the problem ofcytotoxicity, reproduction reaction of acyl CoA which is a coenzymeessential for the reaction is also able to be carried out in the samereaction solution system and consumed amount of the coenzyme drasticallyreduces whereby sphingoid bases are able to be economically produced.According to the present invention, various sphingoid bases are now ableto be produced at a low cost in a pure form and the use becomessignificantly broad.

Moreover, when a thioester exchange reaction is combined with an invitro polymerization in a production process of PHA, the startingsubstance for the reaction is able to be substituted with an easilysynthesizable thiophenyl ester, reproduction reaction of acyl CoA whichis a coenzyme essential for the reaction is also able to be carried outin the same reaction solution system and consumed amount of the coenzymedrastically reduces whereby various PHAs are able to be economically andefficiently produced on an industrial scale and the use becomessignificantly broad.

1. In a production process of a macromolecular compound using anacyltransferase reaction, in which an acyl group of acyl coenzyme A(acyl CoA) is transferred, wherein the reaction is carried out byproduction and/Pr reproduction of acyl coenzyme A from coenzyme A in areaction system by a chemical thioester exchange reaction with an acylgroup donor which is an acyl ester of a thiol compound; wherein an acylgroup donor, acyl group receptor, coenzyme A and acyltransferase arecontained in the reaction system at the same time, an acyl group of theacyl group donor is transferred to coenzyme A by a chemical thioesterexchange reaction to give an acyl coenzyme A and an acyl group of theacyl coenzyme A is transferred to the acyl group receptor; wherein theacyltransferase is a macromolecular polymerization enzyme and amacromolecular compound is synthesized in a reaction in which an acylgroup donor, acyl group receptor, coenzyme A and acyltransferase arecontained in the reaction system at the same time, an acyl group of theacyl group donor is transferred to coenzyme A by a chemical thioesterexchange reaction to give an acyl coenzyme A and an acyl group of theacyl coenzyme A is transferred to the acyl group receptor; and aproduction process of polyester in which the macromolecular compound ispolyester.
 2. The production process of the polyester according to claim1, wherein the polyester is polyhydroxy alkanoate.
 3. The productionprocess of the polyester according to claim 2, wherein the polyhydroxyalkanoate is poly(3-hydroxy alkanoate).
 4. The production process of thepolyester according to claim 3, wherein the poly(3-hydroxy alkanoate) ispoly(3-hydroxy butyrate).
 5. The production process of the polyesteraccording to claim 1, wherein an acyltransferase reaction is repeatedusing acyl coenzyme A or a product by the acyltransferase reaction as anacyl group receptor whereby the macromolecular compound is produced. 6.The production process of the polyester according to claim 1, whereinthe acyl thioester is acyl ester of aromatic thiol.
 7. The productionprocess of the polyester according to claim 6, wherein the acyl ester ofaromatic thiol is hydroxyalkanoate thiophenyl ester.
 8. The productionprocess of the polyester according to claim 7, wherein thehydroxyalkanoate thiophenyl ester is 3-hydroxyalkanoate thiophenylester.
 9. The production process of the polyester according to claim 8,wherein the 3-hydroxyalkanoate thiophenyl ester is 3-hydroxybutyratethiophenyl ester.
 10. The production process of the polyester accordingto claim 1, wherein the macromolecular polymerization enzyme ispolyhydroxy alkanoate synthase.
 11. The production process of thepolyester according to claim 10, wherein the polyhydroxy alkanoatesynthase is derived from genus Ralstonia.
 12. The production process ofthe polyester according to claim 11, wherein the genus Ralstonia isRalstonia eutropha.
 13. The production process of the polyesteraccording to claim 12, wherein Ralstonia eutropha is Ralstonia eutrophaATCC 17699.